Method for purifying lactoflavin



Patented Apr. 22, 1941 UNITED STATES PATENT, OFFICE METHOD FOR- PURIFYING LACTOFLAVIN No Drawing.

Application March 2, 1938,

Serial No. 193.518

11 Claims.

This invention is-concerned with a method for purifying la ctoflavin' and involves an improvement over the procedure described in copending application Serial No. 61,890, filed February 1, 1936, which application resulted in Patent No. 2,186,314, January 9, 1940.

'We have found that lactoflavin, as contained in natural products is more readily soluble (colto as riboflavin. A convenient source is from a crude lactoflavin concentrate, which concentrate loidal as distinguished from. crystalloidal) than tained in natural products is physically or possibly loosely. chemically combined with certain basic types or, natural products which serve to hold it in solution at higher concentrations than when it exists in free form;

- It is an object of the present invention to provide an improved procedure whereby pure lactoilavin may be separated in crystalline form. It is also an object to provide an improved procedure by which extraneous matter, associated with the lactoflavin in natural products, may be separated from the lactoflavln to an extent making it possible to crystallize the pure lactoflavin from a solution in which the natural product would be soluble. A further object of the invention is to provide a process for obtaining pure crystalline lactoflavin without resorting to the formation of intermediate salts of the product and subsequent decomposition thereof as an integrated step in the process of isolation of this material. It is another object to provide a method which involves crystallizing lactoflavin from the same active solvent used for its initial elution and solution from an inert material with which it is associated. It is also an object to disclose a method of producing pure lactoflavin involving the use of not more than four common solvents or precipitants, namely water, acetone, alcohol and ethylether, certain of which substances may at one time act as precipitants for lactoflavin and at another time as solvents or selective eluting agents. Other objects will become apparent. I

The present invention is particularly concerned with the purification of lact oiiavin and its separation from extraneous matter associated with it in solution in water or in an acetone-water mixture. The original source of the lactoflavln may be from milk or from other materials containing this compound. In using the term lactoflavin it is intended. to include flavins from sources other than milk and s metimes referred may be obtained from milk in acordance with United States Patent No. 2,006,699, issued July 2, 1935, or by some other process. It may also be obtained from whey and'other solutions containing lactoflavin.

In the separation of lactoflavin from products containing it in solution, the lactoflavin may be adsorbed on a suitable adsorbate, .such as fullers earth, and may be separated from that adsorbate by elution with an acetone-water solution as described in the above mentioned oopending application. The adsorption is accomplished in an acid solution, for example, at a pH ranging from about 1 to just under '7, a'preferred range being about4.5 to 5.5. This acidity may be obtained by adding hydrochloric or some other suitable acid. The elution may be accomplished with an acetone-water mixture containing, for example, to 85% acetone and preferably containing acetone in the proportion of about to or 82% of the mixture. tone are used in the acetone-water mixture, such mixtures elute a relatively higher proportion of the inert or extraneous material with the lactoflavin. Likewise when higher percentage concentrationsof acetone are employed (in all cases in the presence of some water) such higher concentrations of acetone are relatively less effective in eluting the lactoflavin.

The proportions of the acetone-water mixture to dry material may vary within wide limits dependent upon the equipment used, time relationships and other factors. Proportions as high as 300 or 400 parts by weight of the mixture and as low as seven parts by weight or less of the mixture to one of dry material have been found I satisfactory. The temperature of the elution is not important except that somewhat greater efficiency is obtained at higher temperatures. With suitable equipment to prevent loss or acetone and volatilization, temperatures above the boiling point of the acetone mixture may be used. These preliminary steps in the preparation of the eluate are merely illustrative, it being obvious that the invention will be useful with solutions of the lactofiavin prepared under other conditions.

As a specific example of the preparation of a solution of lactoflavin, 6000 lbs. 01 'fluid lactoflavin or vitamin concentrate, prepared as described in the patent referred to above and containing from 20 to 28% total solids, is placed in an appropriate vat or tank with a suitable high When lower concentrations of acetrate there is added 50 lbs. of commercial muri-' atic acid (sp. gr. about 1.8). The mixture is agitated to secure uniformity and 150 lbs. of a suitable adsorbant grade of fullers earth is then added. The total mixture is subjected to rapid agitation for a period of about 20 minutes, although the time of agitation is not critical. Also, the temperature of the material is not critical since adsorption may be efilciently carried out at ordinary temperatures or -at temperatures even approaching the boiling point of the mixture. Following the agitation period an amount of water is added sufiicient to bring the total weight, exclusive of the fullers earth up to about 8000 to 8250 lbs. The addition of the extra water at this stage is not vital to the process but its addition is made to reduce the relative viscosity of the mixture and to facilitate settling of the .sus-

pended fullers earth.

Following the addition of the extra amount of water the mixture is allowed to stand, without further agitation, until the fullers earth has settled to adegree to permit decantation of practically all of the fluid. The fluid is decanted and the fullers earth is washed with water a number of times by agitation, settling and decanting, and after the last washing and decanting the fraction containing the solids may be filtered in an ordinary filter press in accordance with usual procedure. The filter cake containing the suspended fullers earth on which is adsorbed the lactoflavin and other impurities may be dried in a tunnel dryer or by any other appropriate means, preferably slowly and with exposure to air, or the wet filter cake may be used directly. In the latter case the moisture or water content of the press cake should be ascertained and account taken of the amount of waterv present in order to regulate the percentage of acetone in the acetonewater mixture to be used in the following steps.

Inthe next step of the process, '12 lbs. of the dry fullers earth 'adsorbate (or an equivalent amount of filter press cake based upon the dry solids content) prepared as described above is placed in an appropriate tank equipped with a high speed agitator. To this quantity of dry material there is added 500 lbs. (about 7 to 1 by weight) of a neutral mixture of water and acetone containing about 80% acetone in the aqueous mixture.

The fullers earth adsorbate and the acetoneaqueous mixture are subjected to violent agitation while heating the mixture to about 50 to 55 C. The agitation is continued for a period of not less than to minutes, although periods of from 1 to 2 hours may be employed. Following the agitationperiod th fullers earth is allowed to settle, which settling or precipitation at this stage is very rapid, leaving a clear yellow or greenish-yellow supernatant liquid. This supernatant liquid containing the lactoflavin is decanted oil and the residual fullers earth is subjected to the same elution method for a successive number of times. It has been ascertained that with the material under consideration from 50 to 60% of the lactoflavin is eluted on the first of the lactoflavin from the fullers earth at this stage. The number of elutions may be materially decreased by using larger volumes of the acetone-water mixture per unit amount of the dry fullers earth adsorbate.

The above procedure is merely illustrative-of a method suitable for producing a solution of lactofiavin with which our invention may be used and it is not intended to limit the invention to the particular procedure or details described for preparing the solution. For instance, the inven tion may be applied to other extracts of the dried adsorbate containing lactoflavin or to other aqueous or acetone-aqueous solutions of the lac- In applying our improved procedure to the separation of lactoflavin from a solution of it prepared as described above, the supernatant liquors from the successive elutions are combined and the acetone distilled ofi. Not only is all acetone removed but the residual water is partially removed at this stage. If, for example, four elutions are made each with 500 lbs. of 8.0% acetone-water mixture there would remain substantially 400 lbs. of water after removal of the acetone. This amount of water is evaporated in a. vacuum pan, to a total quantity of about 6 to 12 pounds. The extent to which the solution is to be concentrated will depend upon various factors including the concentration of lactoflavin and the concentration and nature of the-extraneous matter. For instance, in some cases concentrations to 2 to 4 pounds have been found to be satisfactory while in others the concentration is stopped at 15 or more pounds. Concentrations in which the lactoflavin is present in the proportions of 2 to 3 milligrams per cubic centimeter of solution have been found suitable. This concentration should be to such a degree as to partially precipitate the extraneous impurities but to retain the lactoflavin in solution.

The residual 6 to 12 lbs. (or other quantity) of water containing the lactoflavin from the original 72 lbs. of fullers earth adsorbate, together with any precipitate formed therein, is treated with 95% ethyl alcohol, using sufilcient amount of alcohol "so that the final mixture of treatment. Additional elutions release a progressively lesser amount of lactoflavin. It has been determined however that in practically all cases four successive elutions carried out as described will release from to of the lactoflavin. .If it is desired the elution may be continued until all lactoflavin is released. Economy and practical contingencies dictate the degree of recovery water and alcohol has a 50% alcohol concentration by weight. This treatment with alcohol results in the formation of a precipitate which forms on standing and usually has completely precipitated after a period of 8 to 12 hours. The concentration of alcohol used in this treatment is such as is effective in precipitating colloidal fullers earth. Other materials that will accomplish this separation of the fullers earth without deleterious effect upon the lactofiavin may be used.

The precipitate resulting from the alcohol treatment is filtered oil and the filtrate taken to the next step of our process (the handling of this precipitate for the recovery of the adsorbed or occluded lactofiaviuwhich it contains'will be described later). The alcohol contained in the filtrate is boiled off and the remaining aqueous portion further concentrated under vacuum toa final volume of about 800 to 1000 cc. This aqueous volume now contains the greater portion of the lactofiavin contained on the original 72 lbs. of fullers earth adsorbate.

To this 800-1000 cc. of aqueous solution of impure lac'toflavin there is added pure neutral acetone to give a ratio of about 10 volumes of acetone to 1 volume of the aqueous portion. The mixture is thoroughly agitated and allowed to 2,239,285 stand from ml hour. A gummy residue rapand the supernatant liquid may be decanted or filtered.

The optimum concentrations and quantities of acetone-water mixture in this step depend upon the quantity and character of the extraneous material to be precipitated. Ratio .of 1:5 or 1:15, (waterzacetone) for example, may be employed. Increased concentration of acetone will favor increased precipitation of extraneous matter and will also favor crystallization of lactoflavin at this point. Thus if the concentration of acetone is too high the lactoflavin itself may crystallize out. It is desirable. to avoid this at this step but if it should occur the crystals may be filtered out and added to the solution from which the final crystallization of lactofiavin is accomplished. If the concentration of acetone is too low, the material which it is desired to precipitate would not be completely precipitated and flocculated into a coalescent gummy material.

The combined decanted liquors now holding the lactofiavin in solution ,in an acetone-aqueous mixture is freed from acetone by boiling and the aqueous portion further concentrated to about of brilliant yellow or orange-yellow burr-like masses of the pure material. These crystallization nuclei develop in size and number up to a 7 period of about days.

It is desirable to avoid fast crystallization, a. suitable rate being such that it starts after standing about 18 hours at room temperature and is completed in four to five days. If the solution is allowed to stand for a longer time extraneous matter dissolved in the solution may separate out.

It is to be noted that in this step the acetone serves a function directly the reverse of the function of the acetone and water concentration previously used in the procedures described above.

In this stage of the process the'acetone-water concentration must be such that the acetone constituent acts as an agent initiating and facilitating the crystallization of the lactoilavin while in the preceding step its function is to retain the lactoflavin and precipitate extraneous matter. Also at this stage the water must be present in such relative proportions that certain extraneous material. will be held in solution thus permitting the crystallization of the pure lactofiavin facilitated by the appropriate amount of acetone. Thus the lactoflavin, from which extraneous matter associated therewith has been Following the ether treatment of the aqueous portion, ether is removed by decantation and boiling, and pure neutral acetone is again added to the 600 cc. aqueous portion to give a ratio of about 10 volumes of acetone to 1 of water in the aqueous solution. A gummy residue is again formed on standing for a short period. The supernatant liquor is filtered of! and the gummy residue dissolved in a minimum amount of water (about 500 cc), and this aqueous solution again subjected to treatment with 10:1 acetone. The. gummy residue is removed by filtration and the supernatant liquids combined.

Atthis stage the lactofiavin in the original fullers earth adsorbate is now contained in a 10:1 acetonewater mixture. This mixture is now boiled to remove part of the acetone. The

boiling or removal of the acetone at this stage should proceed with care. the amount of acetone removed being only that amount necessary to bring the acetone content of the mixture down to of acetone by weight, with a specific gravity of the mixture in the neighborhood of 0.875. This 70% acetone-water mixture containing the lactoflavin treated as already described is now on the sides and at the bottom of the container separated by the preceding procedures, may be crystallized from a solution in which lactoflavin. having extraneous material associated with it, would be retained in solution. Certain acetone and water relationships have been given in the specific example but it is apparent that these relationships might vary somewhat, such variance being dependent upon the, exact or relative amount of lactoflavin in proportion to the extraneous matter present.

Following completion of the crystallization period the supernatant liquor is decanted and the crystal crop confined to a small volume in the mother liquor. It has been found most expedient .to centrifuge this crystal mass rather than subject-it to filtration. After removal of the mother liquor by decantation and centrifuging the crystals are washed with pure acetone inappropriate volume and by usual procedures but involving decantation and centrifuging in preference to illtration. The acetone washing is repeatedtwo or three times. Following the washing of the crystals with acetone they are further washed 2 or 3 times with cold alcohol employing the decantation and centrifuging manipulation. Following the washing with alcohol the crystals are further subjected to washing a number of times with ethyl ether employing the decantation and The yield of crystals obtained by the process described will of course depend upon the amount of lactoflavin originally contained in the crude lactoflavin concentrate employed; also the yield of crystals will be dependent upon the number of elutions of the dry adsorbate which practical circumstances may dictate. Y

The yellow or orange-yellow satin or threadlike crystals of lactofialvin obtained by the process described are readily recognized by microscopic examination, melting point, solubility, biological activities and other characteristics. v

If for reasons determined by the operator it should be desired to subjectthe crystals' obtained in a further resolution and crystallization the The washed, dried and solvent-free crystals are re-dissolved in water by continued boiling. The aqueous solution of lactoflavin is slowly cooled but without unusual precaution and upon cooling and standing the lactoflavin readily crystallizes in masses of thread-like or needle-like crystals within a period of 12 to 18 hours, depending upon the initial concentration and the rate of cooling. Ii desired these crystals as obtained from the aqueous solution may be washed with acetone, alcohol, and ether or they may be recovered without washing with all or any of the solvents mentioned, depending upon desires of the operator.

The precipitate obtained as the result of treating the concentrated aqueous solution of crude lactoflavin with 50% alcohol by weight as previously described contains substantial amounts of lactoflavin adsorbed on the fiocculated fullers earth and other flocculated impurities precipitated by the 50% alcohol. In many cases it will be found expedient to recover the lactofiavin adsorbed or occluded in thisprecipitate. Recovery of the lactofiavin from this material may be accomplished for example by returning it to a new lot of fullers earth and reprocessing it with the fullers earth in the same identical manner as already described. or the precipitate may be treated independently of a new lot of fullers earth, using the sequence of steps and P ocedures which have been presented in detail as applicable to fullers earth but with appropriate adjustment of the volume of acetone and water mixtures to conform with the smaller volume of inert material and the relatively larger volume or amount of *lactoflavin which this precipitate carries in proportion to the inert material.

In a further modification oi the process the initial elution of the iullers earth adsorbate with 80% acetone and the next following steps may be carried out as previously described up to the point where the aqueous solution of concentrated lactofiavin is reduced to about 6 to 12 pounds. Instead of treating this concentrated aqueous solution with 50% alcohol the following sequence of procedures may be used. The mass may be further condensed in vacuo to a thick consistency, removed from the influence or vacuum and completely dried in the air or in usual types of drying tunnels. Slow drying of the material in the presence of air is desirabled The dried material is leached or simply boiled or extracted with water which dissolves the lactoflavin irrespective of the form in which it may exist at this stage. Redissolving of the dried material however with water does not put into solution all extraneous matter previously existing in solution prior to the drying of the materiaL. A large proportion of inert matter remains insoluble in water, a

- substantial proportion of this inert material isalso insoluble in acetone, alcohol or ether following the drying operation which has been mentioned. The water extract from which the water insoluble material has been filtered (effective and rapid filtration of the insoluble matter may be accomplished) is condensed to a small volume (about 500 to 600 cc.) and this concentrate is the method necessary for the final preparation of to complete dryness as a means for efiectively removing at the stage mentioned a large proportion of inert matter free or substantially free from lactoflavin by virtue of the fact that the drying operation, particularly in the presence of oxygen, renders certain components of the concentrate insoluble in water thus enabling a simple water extraction or solution of the lactoflavin as a method of removal at the particular stag and prior to proceeding with further details of pure crystalline form of lactoflavin. The alternative method also eliminates from the process as a whole the separate treating of the precipitate formed by treatment with 50% alcohol and it likewise eliminates retreating the alcohol precipitate as suggested in the previous discussion.

Instead of following the procedures described above for treatment of the acetone-water (10:1) precipitation or the alcohol precipitation, these precipitates may be reduced-to complete dryness as described in the modified procedure described above and the lactoflavin may be leached from the dried precipitate with water, the water ex tract being returned at an appropriate place in the operation. In such operations the drying is preferably slow and in the presence of air, whereby concurrent physical and chemical changes (possibly oxidation, polymerization,- condensation and/or dehydration) render the inert extraneous material insoluble in alcohol, acetone, ether and water. These changes are apparently accentuated by heat.

It is obvious that many variations in and other applications of the process described above may be made in utilizing our invention and it is not intended to limit the invention to the particular embodiments described. Various temperatures, times, concentrations, etc. may be more suitable in other particular applications of the invention, as will be recognized by ones skilled in the art. Also, the particular sequence of steps described need not be followed and certain of the steps may be omitted where the results of such steps are not required in the particular treatment or for the particular purpose involved. The terms used in describing the invention have been used in their descriptive sense and not as terms otlimitation and it is intended that all equivalents thereo! be included within the scope of the appended claims.

We claim:

1. In the preparation of crystalline lactoflavin', the steps of concentrating an acetone-water solution oflactoflavin and extraneous matter, separating extraneous matter therefrom by alcohol precipitation and ether extraction, treating an aqueous solution of. the lactoflavin with acetone materials in solution.

2. In the preparation of crystalline lactoflavin, the steps of concentrating an acetone-water solution of lactoflavin and extraneous matter derived from an adsorbate, separating extraneous matter therefrom by alcohol precipitation and ether extraction, treating an aqueous solution of such lactoflavin concentrate with acetone to precipitate extraneous matter, removing the acetone precipitated materials and drying them in air,

. 2,239,285 leaching the dried material with water and combining such water solution with the acetonewater fraction from the acetone precipitation, and concentrating the acetone-water mixture containing lactofiavin to lower the proportion of acetone therein to a degree which permits crystallization of the-lactoflavin while maintainin extraneous materials in solution.

3. In the-preparation oi crystalline lactoflavin, the steps of concentrating an acetone=water solution of lactoflavin and extraneous matter from which extraneous matter capable of being precipitated by alcohol has been removed, treating such aqueous solution of the concentrated lactoflavin with acetone to precipitate extraneous matter, removing the acetone precipitated materials, concentrating the fluid fraction to remove acetone, extracting with ethyl ether and separating the ether traction, removing the ether from the aqueous traction and treating with acetone to precipitate extraneous matter, removing the acetone precipitate matter, and concentrating the acetone-water mixture containing lactoflavin to a degree which permits crystallization of the lactoflavin while maintaining the remaining extraneous materials in solution.

a. Inthe preparation of crystalline lactoflavin, the steps of concentrating an acetone-water solution of lactoflavin and extraneous materials, treating such concentrate with cthylalcohol to precipitate extraneous materials, removing the alcohol precipitate and alcohol, separating the ether extractable matter therefrom, treating the resulting aqueous solution with acetone to precipitate extraneous materials while keeping the lactoflavin in solution, removing the acetone precipitated materials and concentrating the acetoneewater mixture containing lactoflavin to a degree which permits crystallization of the lactoflavin while maintaining extraneous materials in solution.

5. In the preparation of crystalline'lactoflavin, the steps of concentrating an acetone-water solution of lactoflavin derived from and extraneous materials, treating such concentrate with ethyl alcohol to precipitate extraneous terials,

removing the alcohol precipitate d alcohol,

drying the precipitate in air and leaching it with water, separating the leach solution and combining it with the liquid portion from the alcohol precipitation, extracting the solution with ether, treating the mixture with acetone to precipitate extraneous materials while keeping the lacto-= fiavin in solution, removing the acetone precipi tated materials and concentrating the acetonewater mixture 6013171.; lactofiavin to a degree which permits crystallization of the lacto while'maintaining extraneous materials in solution.

6. In the preparation of cryste lactoflavin, the steps of concentrating an acetone water solution of lactofiavin and. extraneous material decontaining about 80% acetone, concentrating the eluate and drying'the concentrate in air, leaching the dried material with water, adding acetone to the separated leach solution to give a concentratron or about 10 parts by volume of acetone and 1 part of water, separating the extraneous matter precipitated thereby, heating to remove the acetone, extracting the liquid with ethyl ether, removing the ether and adding acetone to give a concentration of 10 parts oi acetone and l of water, separating any residue, concentrating the liquid portion until the concentration of acetone in water is about 70% and crystallizing the lactoii'avin from this mixture.

'8. In the preparation of crystalline lactofiavin the steps or eluting an adsorbate cont: lactoflavin with a mixture of acetone and containing about 80% acetone, concentrating the eluate, adding ethyl alcohol to give a concentration or about alcohol, separating solids resulting therefrom, heating the liquid fraction to remove alcohol, adding acetone to the resulting solution to give a concentration of about 10 parts bvvolume of acetone and 1 part of water, separating the extraneous matter precipitated thereby, heating to remove the acetone, extracting the liquid with ethyl ether, removing the ether and adding acetone to give a concentration oi 10 parts of acetone and 1 of water, separating any residue, concentrating the liquid portion until the concentration of acetone in water is about 70% and crystallizing the lactoflavin from this mixture.

9. A method ior separating lactoflavln comprising adsorbing the lactoflavin, eluting the adsorbate with a'relatively large quantity of an acetone-water mixture at slightly elevated ternperature, removing acetone and concentrating, treating the concentrate with alcohol, removing the precipitate, adding acetone to the liquid por tion to give a concentrated acetone-water mixture, separating the precipitate, extracting the liquid with ethyl ether, removing the other from rived irom an adsorbate, drying the concentrate.

in air, leaching the dried concentrate th water, extracting the aqueous solution with ether, treating the resulting solution with acetone to. precipitate extraneous tter, remog the acetone precipitated materials d concentrating the acetone-water n iture conta 11- lactovin to a degree which permits cryst nation at the lactoflavin while maintaining extraneous materials in solution.

7. In the preparation of cryst lactovin the steps of eluting an a containin laci flavin with a mixture oi acetone and water the aqueous portion, subjecting this fraction to a concentrated acetone-water mixture, removing the precipitate, concentrating to remove a por-= tion of the acetone and cooling the concentrated solution to crystallize lactofiavin, washing the crystals with acetone, alcohol and ether and drying them,

a portion of the acetoneand cooling the concern trated solution to crystallize lactofiavin, washing the crystals with acetone, alcohol and ether and drying them and returning the precipitate from the alcohol treatment to the elution step.

ill. it. method for separating lactcflavin comprising adsorbing the lactoflavin, eluting the adsorbate with a relatively large quantity of an acetone-water mixture at slightly elevated temperature, concentrating to remove acetone and evaporating to dryness in the presence of air, leaching the dried concentrate with water, separating the liquid fraction, concentrating it and adding acetone to produce a concentrated acetone-water mixture, separating the precipitate, treating the liquid portion with ethyl ether, re-

moving ether trom the aqueous portion, adding acetone to produce a concentrated acetone-water 

